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Additional file 1. Methods used for in-gel digestion, peptide clean-up and liquid chromatography-mass spectrometry.

The Norwegian Scientific Committee for Food and Environment (VKM) initiated this work to examine the extent to which organisms developed by genome-editing technologies pose new challenges in terms of risk assessment. This report considers whether the risk assessment guidance on genetically modified organisms, developed by the European Food Safety Authority (EFSA), can be applied to evaluate potential risks of organisms developed by genome editing.

The Norwegian Scientific Committee for Food and Environment (VKM) initiated this work to examine the extent to which organisms developed by genome-editing technologies pose new challenges in terms of risk assessment. This report considers whether the risk assessment guidance on genetically modified organisms, developed by the European Food Safety Authority (EFSA), can be applied to evaluate potential risks of organisms developed by genome editing.

Additional file 3: Figure S2. Full-length, unprocessed gel pictures of the gels shown in Fig. 1 and Fig. 2D.

Additional file 2: Figure S1. A plot showing the alignment of a the 70 kb contig constructed from nanopore reads from a yellow-fat individual aligned to the ovine reference genome

The Norwegian Scientific Committee for Food and Environment (VKM) initiated this work to examine the extent to which organisms developed by genome-editing technologies pose new challenges in terms of risk assessment. This report considers whether the risk assessment guidance on genetically modified organisms, developed by the European Food Safety Authority (EFSA), can be applied to evaluate potential risks of organisms developed by genome editing. Background Gene technology has allowed for the transfer of genes between organisms and species, and thereby to design altered genotypes with novel traits, i.e. GMOs. A new paradigm started in the early 2000s with the development of genome-editing techniques. Unlike traditional genetic modification techniques resulting in insertion of foreign DNA fragments at random locations in the genome, the new genome-editing techniques additionally open for a few single nucleotide edits or short insertions/deletions at a targeted site in an organism’s genome. These new techniques can be applied to most types of organisms, including plants, animals and microorganisms of commercial interest. An important question is how the novel, genome-edited organisms should be evaluated with respect to risks to health and the environment. The European Court of Justice decided in 2018 to include genome-edited organisms in the GMO definition and hence in the regulatory system already in place. This implies that all products developed by genome-editing techniques must be risk-assessed within the existing regulatory framework for GMOs. The European and Norwegian regulatory frameworks regulate the production, import and placing on the market of food and feed containing, consisting of or produced from GMOs, as well as the release of GMOs into the environment. The assessment draws on guidance documents originally developed by EFSA for risk assessment of GMOs, which were drawn up mainly to address risks regarding insertion of transgenes. The new genome-editing techniques, however, provide a new continuum of organisms ranging from those only containing a minor genetic alteration to organisms containing insertion or deletion of larger genomic regions. Risk assessment of organisms developed by genome editing The present discourse on how new genome-editing techniques should be regulated lacks an analysis of whether risk assessment methodologies for GMOs are adequate for risk assessment of organisms developed through the use of the new genome-editing techniques. Therefore, this report describes the use of genome-editing techniques in food and feed production and discusses challenges in risk assessment with the regulatory framework. Specifically, this report poses the question as to whether the EFSA guidance documents are sufficient for evaluating risks to health and environment posed by genome-edited plants, animals and microorganisms. To address these questions, the report makes use of case examples relevant for Norway. These examples, intended for food and feed, include oilseed rape with a modified fatty acid profile, herbicide-tolerant and pest-resistant crops, sterile salmon, virus-resistant pigs and hornless cattle. The report considers all aspects of the stepwise approach as described in the EFSA guidance documents. Conclusions The inherent flexibility of the EFSA guidance makes it suitable to cover health and environmental risk assessments of a wide range of organisms with various traits and intended uses. Combined with the embedded case-by-case approach the guidance is applicable to genome-edited organisms. The evaluation of the guidance demonstrates that the parts of the health and environmental risk assessment concerned with novel traits (i.e. the phenotype of the organism) may be fully applied to all categories of genome-edited organisms. ............

Additional file 1: Table S1. Contain information about 6 additional structural variants detected within the 70 kb genomic interval harbouring BCO2 gene.

The red muscle color is one of the main quality parameters of Atlantic salmon (Salmo salar). Unique to salmonids, the color comes from the accumulation of astaxanthin in the muscle, which is strictly obtained from the diet since salmon cannot synthesize it de novo. Despite increasingly higher dietary levels of astaxanthin, this quality trait has been slowly abating over the last decade. The diet plays a central role in the coloration together with the farming environment and genetic background which also affects muscle redness. To help understand the enigma, it is important to know how astaxanthin metabolism works in Atlantic salmon and what may affect it. Previous studies have identified that this trait to be of medium-high heritability (h2 = 0.3-0.5), identified two QTLs (quantitative trait loci) and potential causative genes which may explain differences in astaxanthin metabolism in intestine and liver. The candidate genes included abcg2, bco1, and bco1-like, all of which have been knocked out in individual lines using CRISPR/CAS9. In this thesis the different phenotypes have been characterized in order to prove causality. Analysis of specimens (200g) of each line revealed that some individuals of abcg2KO and bco1KO described modest increase in muscle color compared to the wild type controls, the latter line also revealed some specimens with a very pale muscle color. Knockout of bco1-like, however, had a large positive impact on muscle redness. These results are inline with the effect of the QTLs except for the pale bco1KO individuals. However, it is important to keep in mind that all these are mosaic founder fish with variable degree of knockout efficacy. Hence the KO efficiency was calculated using getPCR and specimens of each line with the highest KO percentage were chosen for further analysis. Formalin fixed mid intestine and liver samples were analyzed for lipid droplet content using fluorescence microscopy. Image analysis of liver showed that there was a statistically significant increase in lipid accumulation for all the KO lines, and that lipid droplet size was also affected. For liver, only abcg2KO fish described steatosis, with an increase in lipid load and droplet size. RNA sequencing and differential gene expression (DEG) analysis was carried out in the intestine and liver samples of the KO fish. In addition, corresponding samples from salmon genetically selected to be either red or pale was included in the analysis. The most important findings from DEG came from abcg2KO mid intestine and bco1KO liver. While the other KO fish showed no changes in gene expression. The abcg2KO mid intestine had 1726 genes showing differential expression with several regulated networks involving cholesterol and steroid biosynthetic processes. In addition, four genes involved in carotenoid metabolism were found moderately upregulated (logFold2 change 1.0-1.3; 1-apoA1, carm1, jun and rdh12). In the liver of the bco1KO, there was upregulation of lipid metabolic processes and metabolism with genes involving hypoxia were regulated both ways. In summary, phenotyping the abcg2, bco1, and bco1-like CRISPR/CAS9 knockouts showed that bco1-like affects the muscle redness, with a considerable increase in color intensity. Furthermore, all investigated genes are influencing in lipid metabolism and KO of abcg2 had the greatest effect. M-AA

Oxygen is a fundamental necessity for the fitness and survival of Atlantic salmon. Hypoxia is reduced access to oxygen and low levels of oxygen in body tissues, which affects the welfare, immune response, and product quality of Atlantic salmon. The severity of hypoxia is dependent on exposure time, the size of the fish, life stage and genetics. In the present study, two genetic lines that were selected for fillet color alone (RED and PALE), were exposed to repeated hypoxia (one, two or three times) and the effects on biometric parameters, welfare, skin color, fillet color and fillet quality were evaluated. The body weight, condition factor, and fillet yield of the PALE genetic line was higher compared to the RED genetic line, and hypoxia had no effect on these biometric parameters. The cardiac somatic index of the RED genetic line was higher compared to the PALE genetic line, and hypoxia had no effect. The operational welfare indicators were all affected by exposure to repeated hypoxia. Most of the indicators were not affected by genetics, however the snout damage score was higher for the RED genetic line compared to the PALE genetic line. The fillet color was redder and the astaxanthin content was higher for the RED genetic line compared to the PALE genetic line, but hypoxia did not negatively affect the fillet color. The skin of NQC got darker and less blue with exposure to repeated hypoxia. The skin color of anterior fillets was not affected by hypoxia. The fillet texture got softer as the fish were exposed to repeated hypoxia. Genetics affect color and biometric traits, and hypoxia negatively affects welfare and darkens the skin color of NQC. There needs to be more research that covers the interaction between heart health and astaxanthin retention. In conclusion, further research is needed to look at the performance and mechanisms behind the differences between the two genetic lines. The astaxanthin retention and disposition could be genetically linked to biometric traits and heart health and could be a potential topic suitable for further research. Oksygen er essensielt for Atlantisk laks sin velferd og overlevelse. Hypoksi er redusert tilgang til oksygen og lave nivåer av oksygen i kroppsvev, som påvirker velferd, immunrespons og produktkvalitet hos Atlantisk laks. Alvorlighetsgraden av hypoksi varierer med hvor lenge fisken er eksponert, fiskestørrelse, livsstadiet og genetikk. I denne studien ble to genetisk ulike linjer som var selektert kun for farge (RED og PALE), eksponert for hypoksi (en, to eller tre ganger) og effekten på biometriske parameter, velferd, hud og fillet farge, og fillet kvalitet ble evaluert. Kroppsvekten, filletvekten, kondisjonsfaktor, filletutbytte og kardiosomatisk indeks for den bleke genetiske linjen (PALE/BLEK) var høyere sammenlignet med den røde genetiske linjen (RØD/RED), og hypoksi hadde ingen effekt på de biometriske parameterne. Alle de operative velferdsindikatorene ble påvirket av eksponering av gjentakende hypoksi. De fleste operative velferds indikatorene ble ikke påvirket av genetikk, bortsett fra den RØDE genetiske linjen som hadde høyere skåring på snuteskade sammenlignet med den BLEKE genetiske linjen. Fillet fargen var rødere for den RØDE linjen sammenlignet med den BLEKE linjen. Skinnfargen på NQC ble mørkere og mindre blå med mer eksponering for hypoksi. Skinnfargen på den fremre fileten ble ikke påvirket av hypoksi. Filetteksturen ble mykere ved eksponering av gjentakende hypoksi. Norwegian University of Life Sciences, AquaGen, FHF M-AA

Proper gene expression relies on control by an abundant number of factors and mechanisms, and the orchestration of these fine-tuned programs is fundamental in cellular functionality. Transcription regulation by RNA polymerase II (RNA Pol II) carboxy-terminal domain (CTD) phosphorylation influence global transcription levels and manufacture the adjustment of final RNA products. Several kinases have been reported to be involved in regulation of these intracellular regulatory pathways by phosphorylation of Pol II CTD, and their functional disturbance is linked transcription mis-regulation and disease. Two of the kinases described are cyclin-dependent kinase 12 (CDK12) and CDK13. Research suggests for a role of CDK12 in phosphorylating CTD Serine 2 (Ser2) for productive elongation, while the evidence of CDK13 in its role as a CTD kinase is undefined. They both appear to possess roles in maintenance of genomic stability, but their exact roles are far from established. We here aimed to elucidate the roles of chromatin-bound CDK12 and CDK13 expressed at endogenous levels. From technical causes, CDK13 was excluded from further analyses. This study consequently focused on CDK12 as a CTD Ser2 and Ser5 kinase. Moreover, we aimed to identify the interactome of chromatin-bound CDK12 to acquire knowledge on its role in transcription and RNA processing events. Heat shock treatment of cells was used as a tool to disturb RNA pol II transcriptional programs, and THZ531 was used to inhibit CDK12/13 catalytic activity. Immunofluorescent analyses reveled changed distribution patterns of RNA Pol II and CDK12-GFP upon heat shock- and THZ531 treatment. CTD Ser2 phosphorylation levels decreased under both treatment methods. Obtained results of CTD Ser5 phosphorylation levels were diverging, hence undecidable. Moreover, mass spectrometry (MS) combined with western blot analyses revealed CDK12 to dissociate from chromatin after 30 minutes heat shock treatment. Bioinformatic analyses of the MS data further revealed mRNA processing to be among the most enriched functional groups of CDK12-GFP bound proteome under non-heat shock conditions. RNA splicing- and transcription regulatory factors, and proteins involved in the mRNA surveillance pathway were among the most enriched terms. Together, our data support previous research demonstrating a decrease in Ser2 phosphorylation state upon CDK12/13 inhibition, as well as CDK12 to be a factor involved in transcription and RNA processing events. Korrekt genekspresjon er avhengig av et stort antall faktorer, og en finjusert balanse av deres mekansimer er avgjørende for cellens funksjon. Transkripsjonsregulering ved RNA Pol II CTD fosforylering påvirker transkripsjon på et globalt nivå og justerer endelig nivå av cellens RNA produkt. Tidligere forskning hevder at flere proteiner, såkalte kinaser, er involvert i transkripsjonsregulering ved fosforylering av Pol II CTD, og svekket funksjonalitet av disse proteinene er knyttet til feilregulering av transkripsjon, og eventuell påfølgende sykdom. To av kinasene som er beskrevet er CDK12 og CDK13. Tidligere studier hevder at CDK12 fosforyliserer CTD Ser2 for å fremme produktiv elongering, mens CDK13 i sin rolle som CTD kinase fortsatt er udefinert. De ser begge ut til å være essensielle for å opprettholde genomisk stabilitet, men deres eksakte roller er til dags dato ikke etablert. Målet med denne oppgaven var å belyse endogent uttrykt CDK12 og CDK13 i sine roller som kromatin-bundne CTD kinaser. CDK13 ble etter tekniske årsaker ekskludert fra videre analyser. Denne studien fokuserte som følger på CDK12 som CTD Ser2 og Ser5 kinase. Videre var målet å identifisere proteiner som naturlig interagerer med kromatin-bundet CDK12 for å oppnå kunnskap om dette proteinets rolle i transkripsjon og RNA-prosessering. Varmebehandling av celler ble brukt som et verktøy for å forstyrre RNA Pol II under transkripsjonen, og THZ531 ble brukt for å forstyrre CDK12/13 katalytisk aktivitet. Immunofluorescerende analyser avslørte endrede fordelingsmønster av RNA Pol II og CDK12-GFP som følger av varme- og THZ531 behandling. Fluorescerende nivåer av CTD Ser2 sank under begge behandlingsmetodene, mens nivåene av CTD Ser5 var tvetydige. MS kombinert med western blot analyse avslørte at CDK12 frigjøres fra kromatin etter 30 minutter med varmebehandling. Bioinformatiske analyser demonstrerte videre at mRNA-prosesserende faktorer var blant de største funksjonelle gruppene som under normale forhold er bundet til CDK12-GPP. Faktorer involvert i RNA spleising- og transkripsjonsregulering, samt i kvalitetskontroll av mRNA, var blant de mest interessante funnene. Våre data støtter tidligere forskning som viser en nedgang i CTD Ser2 fosfoyleringsnivå etter CDK12/13-inhibering, samt at CDK12 er involvert i transkripsjon og prosessering av RNA. M-BIOTEK